A disintegrin and metalloproteinase 15-mediated glycocalyx shedding contributes to vascular leakage during inflammation

Abstract

Aims: Endothelial hyperpermeability exacerbates multiple organ damage during inflammation or infection. The endothelial glycocalyx, a protective matrix covering the luminal surface of endothelial cells (ECs), undergoes enzymatic shedding during inflammation, contributing to barrier hyperpermeability. A disintegrin and metalloproteinase 15 (ADAM15) is a sheddase capable of cleaving the ectodomains of membrane-bound molecules. Herein, we tested whether and how ADAM15 is involved in glycocalyx shedding and vascular leakage during sepsis.

Methods and results: Dextran-150kD exclusion assay revealed lipopolysaccharide (LPS) significantly reduced glycocalyx thickness in mouse cremaster microvessels. Consistently, shedding products of glycocalyx constituents, including CD44 ectodomain, were detected with an increased plasma level after cecal ligation and puncture (CLP)-induced sepsis. The direct effects of CD44 ectodomain on endothelial barrier function were evaluated, which revealed CD44 ectodomain dose-dependently reduced transendothelial electrical resistance (TER) and caused cell-cell adherens junction disorganization. Furthermore, we examined the role of ADAM15 in CD44 cleavage and glycocalyx shedding. An in vitro cleavage assay coupled with liquid chromatography-tandem mass spectrometry confirmed ADAM15 cleaved CD44 at His235-Thr236 bond. In ECs with ADAM15 knockdown, LPS-induced CD44 cleavage and TER reduction were greatly attenuated, whereas, ADAM15 overexpression exacerbated CD44 cleavage and TER response to LPS. Consistently, ADAM15 knockout in mice attenuated CLP-induced increase in plasma CD44. Intravital and electron microscopic images revealed ADAM15 deficiency prevented LPS-induced glycocalyx injury in cremaster and pulmonary microvasculatures. Functionally, ADAM15-/- mice with better-preserved glycocalyx exhibited resistance to LPS-induced vascular leakage, as evidenced by reduced albumin extravasation in pulmonary and mesenteric vessels. Importantly, in intact, functionally vital human lungs, perfusion of LPS induced a significant up-regulation of ADAM15, accompanied by elevated CD44 in the effluent and increased vascular permeability to albumin.

Conclusion: Together, our data support the critical role of ADAM15 in mediating vascular barrier dysfunction during inflammation. Its mechanisms of action involve CD44 shedding and endothelial glycocalyx injury.

Figure 1

LPS causes glycocalyx shedding and microvascular leakage. (A, B) Measurement of mouse microvascular glycocalyx thickness. Mouse cremaster microvascular glycocalyx was determined using FITC-dextran (150 kD) exclusion assay 24 h after LPS injection. (A) Representative bright-field and fluorescent images of mouse cremaster microvessels. Scale bar = 20 μm. (B) Quantification of the glycocalyx thickness (mean ± standard deviation). n = 5 independent animals in each group, each dot represents the average of three measurements in one microvessel. *P < 0.05. (C) The effects of LPS on the plasma level of HA. Mouse plasma was collected 24 h after LPS injection (mean ± standard error of the mean (SEM), n = 6). *P < 0.05. (D) Evans Blue extravasation showing plasma protein leakage in mouse lung. Left lung lobes from vehicle or LPS-treated mice were scanned with infrared imaging system. Signal intensity is proportional to the amount of Evans Blue-bound albumin extravasated into the extravascular space. n = 4. *P < 0.05.

Figure 2

CD44 ectodomain exhibits barrier disruptive effects. (A) The plasma level of soluble CD44 in mice subjected to sham or CLP induced-sepsis (mean ± SEM, n = 5). (B) Time- and does-dependent TER tracing showing the effects of CD44 ectodomain on endothelial barrier function. Values were normalized to their baseline (t = 0). Solid line represents the mean resistance, and shadow represents standard error. Embedded bar graph indicates maximal TER drop (mean ± SEM, n = 3). (C) Representative confocal images of endothelial adherens junctions upon vehicle or CD44 ectodomain (10 μg) treatment. Scale bar = 20 μm. (D) Discontinuity of VE-cadherin and β-catenin at cell–cell junctions. The number of discontinuous adherens junction positive-stained regions was quantified using Imaris software (mean ± SEM, n = 3). Each dot represents the average value from five fields of view. (E, F) The effects of CD44 ectodomain on tyrosine phosphorylation of VE-cadherin. CD44 ectodomain was applied to HUVEC monolayer and incubated for 5 min. Cell lysate was immunoblotted for phosphorylated VE-cadherin (p-VE-cadherin) and total VE-cadherin (VE-cadherin). (E) Representative western blot images. (F) The ratio of densitometry value of p-VE-cadherin to total VE-cadherin (mean ± SEM, n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001 vs. vehicle control.

Figure 3

Recombinant ADAM15 cleaves CD44 at His^235-Thr²³⁶ bond. (A) Western blot showing ADAM15 cleaves CD44 in a dose-dependent manner. Recombinant ADAM15 was incubated with CD44 at 37°C for 6 h. The protein mixture was immunoblotted for ADAM15 and CD44. (B) Densitometry analysis of full-length CD44 and cleaved CD44 bands (mean ± SEM, n = 3). *P < 0.05 vs. control. (C) Schematic diagram of the CD44 amino acid sequence and cleavage site. Cleaved-CD44 band was digested by trypsin, sequenced via LC-MS/MS and matched with the full-length CD44 sequence (matched amino acids were in red). The C-terminal amino acid His²³⁵ (in black box) of the non-tryptic peptide was considered the cleavage site.

Figure 4

ADAM15 expression regulates CD44 ectodomain shedding. (A, B) The effects of ADAM15 knockdown on the shedding of CD44 ectodomain. (A) ADAM15 expression on cell membrane after LPS (1 μg/mL, 24 h) treatment in HUVECs with or without ADAM15 siRNA knockdown. Mean ± SEM, n = 3. Na/K ATPase serves as membrane fraction loading control. *P < 0.05 vs. vehicle + control siRNA, # P < 0.05 vs. LPS + control siRNA. (C, D) The effects of ADAM15 overexpression on CD44 ectodomain shedding. (C) Verification of ADAM15 overexpression (mean ± SEM, n = 3). Na/K ATPase = membrane fraction loading control. (D) The effects of ADAM15 overexpression on soluble CD44 level in HUVEC conditioned medium (mean ± SEM, n = 3). *P < 0.05 vs. control plasmid.

Figure 5

ADAM15^−/− mice are resistance to sepsis-induced glycocalyx degradation and vascular leakage. (A) The plasma level of soluble CD44 in wild-type and ADAM15^−/− mice after sepsis (mean ± SEM, n = 6). *P < 0.05. (B, C) The effects of ADAM15 deficiency on sepsis-induced glycocalyx shedding. (B) Representative images of mouse cremaster vessels in wild-type and ADAM15^−/− mice subjected to LPS injection. Scale bar = 20 μm. (C) Quantification of the glycocalyx thickness (mean ± SD, n = 5 animals). Each dot represents the average of three measurements in one microvessel; *P < 0.05. (D) Electron microscopy images of pulmonary microvascular glycocalyx in wild-type and ADAM15^−/− mice subjected to LPS challenge. Arrows indicated the endothelial glycocalyx. Scale bar = 1 μm. (E, F) The impacts of ADAM15 deficiency on LPS-induced vascular hyperpermeability. (E) Pulmonary vascular permeability was determined by Evans Blue extravasation assay 24 h after LPS injection. Evans Blue bound-albumin extravasation was quantified using LI-COR Odyssey Clx (mean ± SEM, n = 4). *P < 0.05 vs. vehicle + wild-type, #P < 0.05 vs. LPS + wild-type. (F) Intravital microscopy images showing FITC-albumin leakage from mesenteric microvessels (n = 4). Scale bar = 100 μm.

Figure 6

Ex vivo human lung subjected to LPS treatment exhibits increased glycocalyx damage and vascular hyperpermeability. (A) Ex vivo human lung perfusion system. Human lungs were ventilated and perfused with 0.25 mg/mL LPS for 6 h. (B) Western blot showing the expression of ADAM15 in human lung tissue with or without LPS treatment. GAPDH serves as loading control (mean ± SEM, n = 3). (C) The level of soluble CD44 level in the perfusion effluent (mean ± SEM, n = 3). (D) Circulating HA level in the perfusion effluent (mean ± SEM, n = 3). (E) Evans Blue extravasation assay showing human pulmonary microvascular permeability. n = 3. *P < 0.05.

Figure 7

Schematic diagram showing ADAM15-induced glycocalyx damage contributes to vascular barrier disruption during inflammation. Under normal condition, the endothelial glycocalyx and the adherens junction at endothelial cell–cell contacts act collectively to prevent the transvascular leakage of plasma proteins. However, upon inflammation, up-regulated ADAM15 mediates the cleavage of CD44 via its metalloproteinase domain, resulting in the disruption of glycocalyx structural integrity. Reduced glycocalyx coverage impairs its barrier property against macromolecular leakage and increases the exposure of ECs to inflammatory mediators in the circulation. The cleaved products of glycocalyx (CD44 ectodomain and low-molecular-weight HA) cause adherens junction disorganization, which allows more plasma protein leakage into the interstitial space.